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-   -   Template quantification using UV absorbance? (http://www.molecularstation.com/forum/agarose-gel-electrophoresis-forum/72905-template-quantification-using-uv-absorbance.html)

nebulus 05-02-2010 07:30 AM

Template quantification using UV absorbance?
 
I am trying to prepare my DNA template for sequencing and I am required to provide my template concentration.

From what I have read I shouldn't be able to accurately determine my PCR product concentration via spectroscopy? I tried quantifying my gel purified extract on a nanodrop-1000 and got a reading of 0.1ng/ul.

Instead I should use a dna concentration ladder to run next ti my samples in my gel.

Is this correct? I am new at this.


Thanks

Drekketh 05-10-2010 11:47 AM

Re: Template quantification using UV absorbance?
 
You are both correct. If your Nanodrop apparatus is equilibrated nicely is very sensitive in measuring even PCR products. By all means it is also correct to run a weighted DDNA ladder next your sample and determine approximately the DNA concentration by the intensity of the band.

Multiplexion 02-22-2013 05:18 PM

Re: Template quantification using UV absorbance?
 
You can try the following:

Load serial 1:2 dilutions ranging from 4 ÁL to 0.125 ÁL of each amplimer and the Smart-Ladder DNA quantification marker on a 2 % agarose gel. Take digital photographs and measure the concentrations using Image Quant software.

Intensities of amplimer bands can be compared with those of the quantification marker and, thus, concentrations can be computed.


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